Kidneys are bean-shaped organs that serve as a biological filter removing metabolic waste from blood to the urinary bladder. The basic structural and functional unit of kidney is a nephron, the main function of which is to regulate the concentration of water and soluble substances in blood, reabsorbing the vital elements and excreting the rest as urine. In humans, a normal kidney contains 800,000 to 1.5 million nephrons. The adequate kidney function is imperative for survival: kidneys control level of electrolytes in blood, regulate blood volume, blood pressure and blood pH.
Bacterial infections, accumulation of toxic materials and auto-immune diseases may cause inflammatory disease of kidneys, also called nephritis. The symptoms of nephritis are a rise in blood pressure, back pain, edema and fatigue. Albumin and other serum proteins in increasing quantities pass into urine, creating a condition called proteinuria. If the condition continues to deteriorate, uremia or renal failure develops. Renal failure leads to accumulation of high concentrations of metabolic waste in the blood and eventually causes death. The conventional medical treatment is the removal of this waste by filtering blood with a dialysis machine, also called an artificial kidney. When the condition deteriorates still further, transplantation of a kidney is the last and the only option.
Chronic kidney disease has several underlying reasons and involves the glomeruli, tubules or interstitial tissue surrounding the glomeruli and tubules. The glomerulus is a network (tuft) of capillaries that performs the first step of blood filtration. The renal tubule is the part of nephron containing the fluid filtered through the glomerulus. Glomerulonephritis (GN) is inflammation of glomeruli. GN may lead to serious kidney damage and, in some patients, kidney failure. The causes of glomerulonephritis are complex and diverse, some have a genetic basis and others are associated with systemic diseases. While treatment is available for some types of GN, many traditional therapies are toxic, non-specific and have the potential for major side effects. Some GN subtypes do not respond to any therapies. Worldwide, GN is the most common single cause of end-stage renal disease (ESRD).
There are more than 20 million Americans with chronic kidney disease and 50,000 of them die annually.
Though chronic autoimmune disorders such as systemic lupus erythematosus affect a significant percentage of the human population and strongly diminish the quality of life and life expectancy, the molecular mechanisms of those diseases are still poorly understood, hindering the development of novel treatment strategies. Autoimmune diseases are caused by disturbed recognition of foreign and self-antigens, leading to the emergence of auto-reactive T-cells (so-called immunization phase). T-cells are a major regulator of the inflammatory cascade. The auto-reactive T-cells then trigger the second (so-called effector) phase of the disease which is characterized by activation of the immune cells that cause immune-mediated damage to host tissues.
For a long time, neutrophils have been neglected as potential players in the development of autoimmune diseases. However, a significant amount of new experimental data suggests that neutrophils play an important role in both the immunization and the effector phase of autoimmune diseases. Taken together, neutrophils should be considered as one of the most important cell types in autoimmune diseases and a suitable target for treatment of those diseases.
Neutrophils are the most common type of white blood cell in human body and are the first line of attack against invaders. In a person with a healthy immune system, the white blood cells gather at an infected or injured site in the body and produce chemical substances that help fight off the infection. These substances increase inflammatory reaction and attack invaders, causing some collateral injury to healthy tissue. Usually the immune system is capable of producing additional substances that make the inflammatory process self-contained and limited in time.
Inflammation in a healthy individual usually signals that the immune system is responding appropriately to harmful invaders, damaged cells, irritants, or injury. But, in case of autoimmune diseases, due to faulty interactions between blood and other immune system cells, the inflammation increases. Attracted by cytokines (messenger molecules) expressed by activated endothelium and other residential cells in inflamed tissues, neutrophils, using “roll”, “stop” and “exit” mechanism leave blood vessels and congregate at a focus of infection. Migrated into the kidney, neutrophils release their own cytokines, which in turn activate several other cell types and amplify inflammatory response.
In addition to recruiting and activating other cells of the immune system, neutrophils play a key role in the front-line defense against invading pathogens. Neutrophils have three methods for directly attacking micro-organisms: phagocytosis, release of soluble anti-microbials substances, and generation of neutrophil extracellular traps (NETs). NETs are networks of extracellular fibers, primarily composed of DNA from neutrophils, which bind and kill extracellular pathogens while minimizing damage to the host cells. NETs may also have a deleterious effect on the host. It is believed that excessive expression of extracellular histone complexes from DNA combined with their slow clearing plays an important role in the development of autoimmune kidney diseases, particular in lupus.
There is a need in developing of novel therapies that inhibit recruitment of neutrophils from blood vessels and deactivate neutrophils that already aggregated in kidneys.
The autoimmune diseases cause significant numbers of patients that progress to ESRD. But the most important disease responsible for the highest number of patients with ESRD is diabetic nephropathy. The growing epidemic of obesity-related insulin resistance and the difficulties in managing diabetes have made diabetic nephropathy the major single cause of kidney disease in the developed world.
Despite current clinical interventions involving tight glycemic and blood pressure control, diabetic nephropathy progresses in most patients with a significant proportion reaching end-stage renal failure. In addition, the development of diabetic nephropathy exacerbates cardiovascular disease, which leads to increased morbidity and mortality. Given the limited renal protection with current treatments, it is critical that alternative therapeutic approaches are developed to protect diabetic patients from diabetic kidney disease.
Recent studies have identified macrophage-mediated injury as an important component in the development of diabetic nephropathy that is not addressed by current therapies. Further evidence has shown that macrophages are the major immune cells infiltrating the kidney in type 1 and type 2 diabetes, and that they contribute to the development of renal injury. In view of this new evidence, diabetic nephropathy has been reclassified as a chronic inflammatory disease which is triggered and maintained by metabolic disturbances of diabetes mellitus.
Elements of the diabetic milieu activate the vascular endothelium, inducing increased expression of cell adhesion molecules (ICAM-1/VCAM-1) that adhere to circulating blood monocytes. Using the “roll”, “stop” and “exit” sequence the blood monocytes migrate from the blood vessels to the kidney, where they differentiate into macrophages. Glomerular podocytes, mesangial cells and tubular epithelial cells are also stimulated by the diabetic milieu. They additionally secrete chemokines (MCP-1/OPN) that facilitate transendothelial and intrarenal monocytes/macrophage migration. Also, the diabetic renal parenchymal cells produce colony stimulating factor-1 (CSF-1) which induces local proliferation of macrophages that also contributes to the accumulation of macrophages in diabetic kidneys.
Activated macrophages release reactive oxygen species and pro-inflammatory cytokines, which cause injury to podocytes, interstitial and tubular cells. These macrophages also can secrete profibrotic cytokines that induce mesangial and fibroblast proliferation and development of fibrosis. This ongoing renal injury and fibrosis promotes the progression of diabetic nephropathy.
Renal interstitial fibrosis (RIF) is the common pathological process of chronic kidney diseases leading inevitably to renal function deterioration. RIF and preceding epithelial-mesenchymal transition (EMT) are commonly triggered in diabetic kidney by an early occurring renal inflammation. However, an effective approach to prevent EMT and RIF is still lacking.
Adenosine A2a receptor recently emerged as a potent inflammation regulator. Adenosine activation of A2a receptors suppresses the EMT process and protects kidneys against RIF. Experimental studies suggest that activation of A2a significantly suppresses the deposition of collagen types I and III thus inhibiting the EMT progress. As a result, activation of A2a effectively alleviates EMT and RIF, suggesting A2a receptors as a potential therapeutic target for treatment of RIF.
Development of therapeutic strategies for treatment of macrophage-mediated injury in kidney that selectively target mechanisms of macrophage recruitment, proliferation and activation seems to be an attractive choice in comparison with general immunosuppression.
Now it is commonly accepted that macrophages play a critical role in the development of diabetic nephropathy. Current therapies, however, are unable to prevent the progressive renal damage caused by these inflammatory cells. There is an urgent need for novel anti-inflammation and immuno-suppressive therapies aimed at reducing macrophage recruitment, accumulation and activation in diabetic kidneys.
Adenosine is a purine nucleoside generated by metabolically stressed or inflamed tissues that is recognized as a major endogenous anti-inflammatory regulator. Under normal conditions, adenosine is continuously released from cells as a product of ATP degradation. Adenosine concentration in extracellular space is controlled by an enzyme called adenosine deaminase (ADA) which breaks it down and keeps the concentration level in low-micromolar to high-nanomolar range.
However, during conditions of stress, such as hypoxia during inflammation, levels of extracellular adenosine rise dramatically (up to 200-fold). This is partly due to increased production of AMP in hypoxic conditions, but substantial amounts of adenosine are also produced by the sequential dephosphorylation of adenine nucleotides released from platelets and hematopoietic cells, as well as damaged cells.
Adenosine regulates the function of the innate and adaptive immune systems through targeting virtually every cell type that is involved in orchestrating the immune/inflammatory response. Of the four adenosine receptors (A1, A2a, A1b, A3), A2a receptors have taken center stage as the primary anti-inflammatory effectors of extracellular adenosine. This broad, anti-inflammatory effect of A2a receptor activation is a result of the predominant expression of A2a receptors on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells, eosinophils, epithelial cells, as well as lymphocytes, NK cells, and NKT cells. A2a receptors play a critical role in controlling leukocyte trafficking by suppressing release of cytokines that induce production of adhesion molecules (ICAM-1/VCAM-1) and promote the “roll”, “stop” and “exit” mechanism bringing neutrophils and monocytes/macrophages from blood vessels into tissues.
A2a receptor activation inhibits early and late events occurring during an immune response, which include immune cell trafficking, immune cell proliferation, proinflammatory cytokine production, and cytotoxicity. In late stage of inflammation in addition to limiting inflammation, A2a receptors participate in tissue remodeling and restoration. Consistent with their multifaceted, immunoregulatory action on immune cells, A2a receptors have been shown to impact the course of a wide spectrum of ischemic, autoimmune, infectious, and allergic diseases.
A2a receptors are found in all parenchymal cells in kidney: glomerular endothelium, podocytes, tubular endothelium and mesangial cells. Adenosine A2a receptor activation was recently shown to be renoprotective in diabetic nephropathy. Activated A2a receptors protect kidneys from diabetic nephropathy through actions on hematopoietic and kidney-derived cells. Podocytes contribute to the maintenance of the glomerular filtration barrier and abnormalities of podocyte structure and function lead to a number of glomerular diseases. Activation of adenosine A2a receptors preserves the structure and function of podocytes and leads to a reduction of proteinuria and preservation of glomerular function.
Macrophages play a critical role in immune response against pathogenic invaders. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role and can decrease immune reactions through the release of cytokines. Macrophages that encourage inflammation are called M1 macrophages, whereas those that decrease inflammation and encourage tissue repair are called M2 macrophages. This difference is reflected in their metabolism, where macrophages have the unique ability to metabolize one amino acid, arginine, to either a “killer” molecule (nitric oxide) or a “repair” molecule (ornithine).
Macrophages morph in phenotypes M1 or M2 depending on the environment in which they are activated. In the presence of cytokines such as IL-12 and IL-23 from T helper-1 cells that orchestrate initial stage of immune response, the “classically” activated macrophages are pro-inflammatory M1 type, whereas at the final stage of inflammation in presence of cytokine IL-10 and TGF-β from T helper-2 cells, macrophages become “alternatively” activated into anti-inflammation type M2, that promote tissue restoration. The same macrophage that was a pro-inflammatory M1 type at the beginning of inflammation can be re-activated into an anti-inflammatory M2 type at the final stage of the inflammation.
Consistent with its generally restorative function in tissues, A2a receptor activation has been repeatedly shown to have effects that prevent excessive classical macrophage activation thereby resulting in tissue protection. In contrast to the suppressive effect of adenosine on the production of proinflammatory mediators, adenosine augments production of anti-inflammatory cytokine IL-10 that promotes activation of macrophages into M2 type.
Thus, in addition to deactivating classically activated macrophages, A2a receptor signaling changes macrophage metabolism and enables switching their phenotype to alternative type M2 that participates in tissue restoration.
The protective effect of stimulation of A2a receptors was proven to correlate with decreased expression of adhesion molecules (ICAM-1/VCAM-1) and reduction of transmigration of neutrophil and monocytes/macrophages into the kidney. In addition, the renal microvasculature responds to A2a receptors stimulation by vasodilation that results in increased blood flow in the renal microcirculation and contributes to the renal protection.
Pulsed Electromagnetic Field Therapy (PEMF) is a new non-invasive method of treatment of numerous medical conditions related to injuries and inflammations of different tissues: bones, cartilages, soft and neurological tissues. For centuries it was a common knowledge that the natural wound healing involves generation of endogenous electric fields. Recently it has been discovered that the endogenous electric fields control also the processes of remodeling and healing bones and cartilages.
In PEMF therapy the electric field is carried into the treatment zone by a pulsed magnetic field produced by electromagnetic coils from outside the body. A PEMF system applies a series of magnetic pulses to injured tissue where each magnetic pulse induces an electrical signal that stimulates cellular anti-inflammatory and anabolic activities. PEMF therapy reduces pain associated with inflammation by suppressing production of pain mediator prostaglandin E2 and accelerates natural healing of tissues. Multiple studies have demonstrated effectiveness and safety of PEMF therapy in suppressing inflammation.
Recently it has been established by Varani et al. that the anti-inflammation mechanism of action of PEMF on a cell is due to its ability to increase the concentration of receptors A2a on the cell membrane. PEMF stimulation increases the number of active A2a receptors on the cell membrane by creating a conformational change of their protein and making them active and available for binding with adenosine ligand. The signal to the cell and the biological response of the cell's machinery depends on both the concentration of ligands in extracellular space and the concentration of receptors on the cell membrane. In other words, the magnitude of biological response of the cell depends on the product of these two concentrations. As a result, the same response can be achieved by two different ways: by changing concentration of adenosine around the cell or by changing concentration of the receptors on the cell membrane. The essence of the discovery of Varani et al. is that the adenosine signaling pathway can be up-regulated without changing extracellular adenosine concentration. It can be achieved by PEMF stimulation alone.
According to the experimental data, the A2a receptors can be up-regulated by the pulsed electric fields with amplitude above 50 μV/cm. In the environment rich in extracellular adenosine, which is always the case with inflamed or stressed tissues, the up-regulation of A2a receptors leads to significant amplification of adenosine signaling. PEMF stimulation triggers the same physiological response of the cell as an increase in concentration of adenosine or another A2a agonist in the extracellular space. In either case, the magnitude of signal from A2a receptors to the cellular machinery increases as well as the downstream effects of the A2a signaling.
PEMF stimulation can affect a wide variety of cells that express A2a receptors, including T cells, macrophages, neutrophils and other lymphocytes. All parenchymal kidney cells carry A2a receptors and can be stimulated by PEMF. A2a receptors stimulated by PEMF are able to inhibit multiple processes occurring during an immune response, including immune cell trafficking and proliferation, pro-inflammatory cytokine production and cytotoxicity. In addition to limiting inflammation, A2a receptors participate in tissue remodeling and repair. A2a receptors have been shown to impact the course of autoimmune, infectious, and allergic diseases.
PEMF stimulation of A2a receptors generates immunosuppressive action by inhibiting overreactive immune cells, thereby protecting tissues from collateral inflammatory damage. PEMF stimulation of A2a receptors provides a novel regulatory tool for immune/inflammatory diseases of various organs, including kidney. They can be a critical part of the physiological negative feedback that limits local inflammatory responses. Increased by PEMF stimulation, A2a signaling inhibits development of cytotoxicity and cytokine-producing activity in T-cells. Stimulated by PEMF A2a receptors in autoreactive T-cells generate strong immunosuppressive action that reduces chronic inflammation and subsequent damage to the affected organ.
Upregulation of adenosine A2a signaling by PEMF in hematopoietic and renal parenchymal cells results in cascades of actions. It can down-regulate recruitment of inflammatory leucocytes from blood vessels by acting on the vascular epithelial cell and disrupting the “roll”, “stop” and “exit” mechanism. Via A2a receptors signaling PEMF promotes changing phenotypes of macrophages from pro-inflammatory type M1 to anti-inflammatory type M2, stimulates resolution of inflammation and tissue restoration. It promotes improvement of function of podocytes, reduction of proteinuria, and down-regulation of expression of pro-inflammatory cytokines in mesangial cells—the major source of inflammation in diabetic kidney. Also it inhibits renal interstitial fibrosis (RIF) by suppressing the epithelial-mesenchymal transition (EMT).
These outstanding anti-inflammation and immuno-suppressive actions of A2a receptors attracted attention of many researchers as a potential basis for development of new drugs for treatment of various inflammatory diseases. Unfortunately, free adenosine has a very short half-life time in plasma, about 10 seconds, which severely limits its usage as a systemic drug. However, a number of agonists with high selectivity to A2a receptors and long half-life in plasma have been developed recently, keeping the hope of developing these drugs alive.
It should be noticed that all new knowledge recently accumulated about A2a effects on the inflamed tissues came mainly from in vitro experiments and animal trials. Unfortunately, in human clinical trials, a big problem with adenosine agonists has been encountered—a strong hemodynamic effect.
A2a receptors are abundantly expressed in vascular endothelium and play a significant role in regulating blood flow throughout the body. Systemic activation of A2a receptors leads to vasodilation in the whole vascular bed and increases blood flow, which, in turn, creates a significant reactive drop in blood pressure, increase in heart rate and cardiac index. These hemodynamic side effects limit systemic dosing of A2a agonists to the level at which they are no longer effective at resolution of inflammation. This problem has contributed to the failure of several A2a agonists. Examples of discontinued A2a agonists trials include GW328267X from GlaxoSmithKline PLC and UK-432097 from Pfizer Inc. Several A2a agonists, though, are still in clinical trials for inflammation-related indications.
The anti-inflammation and immuno-suppressive actions of A2a receptors can be employed by using PEMF stimulation of the affected areas. For more than thirty years of experimental and clinical use of PEMF stimulation for different tissues there were no noticed side effects. PEMF stimulation boosts activity of A2a receptors locally; it does not increase the concentration of adenosine around the cells, but instead, increases A2a concentration on the cellular membranes.
Local PEMF stimulation effects inflamed tissues only, where the concentration of adenosine is high, up to 200-fold of the base line. As has been mentioned before, under normal physiological conditions concentration of adenosine in tissues is low and PEMF stimulation effects are minimal. This is the major reason why PEMF therapy does not have side effects.
Stress response is one of the most important biological reactions to a wide variety of unfavorable physiological and environmental conditions. It is a part of cell's own repair system that is evolutionary conserved and universally expressed from bacteria to humans. One of the first cellular reactions on stress is rapid generation of so called heat shock proteins (HSPs). Heat-shock proteins play numerous roles in cell function, including modulating protein activity by changing protein conformation, promoting multi-protein complex assembly/disassembly, regulating protein degradation within the proteasome pathway, facilitating protein translocation across organelle membranes, and ensuring proper folding of nascent polypeptide chains during protein translation.
When cells are overstressed, the common response is to undergo cell death by one of two pathways, either ‘necrosis’ or ‘apoptosis’. Recently, both routes to cell death have been revealed to share similar mechanisms, with heat shock proteins and their cofactors responsible for inhibiting both apoptotic and necrotic pathways. So, the effective biological function of HSPs is to preserve cell survival by maintaining the vital functions of proteins.
In practical terms, HSPs can be induced by local thermal stimulation with temperatures 40-43 degrees C. for 10-30 minutes.
Deep thermal stimulation can be achieved by ultrasound, high radiofrequency and microwave diathermia devices. Also, kidneys can be stimulated as a part of the whole body thermal stimulation in a hot bath, sauna and steam room.
After thermal stimulation, intracellular concentration of HSPs rapidly grows to several fold level at 24 hours and returns to the basal level after 48-72 hours. Intracellular buildup of HSPs is triggered by activation of Heat Shock Factor (HSF), whereas HSP concentration is controlled by enzyme adenosine deaminase (ADA) that degrades HSPs. Luckily, the activity of ADA is inhibited by PEMF stimulation, so, as a result, PEMF helps to keep the HSPs concentration high.
It is known from animal studies that HSP72 inhibits proliferation and apoptosis in tubular cells and diminishes accumulation of fibroblasts and collagen in renal parenchyma, thus slowing the process of fibrosis. It was also observed that HSP70 exerts strong cytoprotection of mesangial cells from oxidative injury in experiments with ischemic reperfusion. Overall, a significant body of evidence suggests that HSPs delay the progression of chronic kidney disease (CKD) by the anti-apoptotic activity and cytoprotection.
Koga et al. in the article “Mild electrical stimulation and heat shock ameliorates progressive proteinuria and renal inflammation in mouse model of Alport syndrome” demonstrated that combination of electrical and heat stimulation provides anti-proteinuric and anti-inflammatory effects on Alport mice through multiple signaling pathways via podocytic activation of Akt (protein kinase B) and induction of HSP72. The authors suggest a new therapeutic strategy to decelerate the progression of Alport syndrome by applying combined electrical and heat stimulation. In the experiment described in the article, the electrical stimulation was provided by a pulsed direct electric current applied to the experimental animals via electrodes attached to the skin. The thermal stimulation was delivered from the same electrodes.
In U.S. Pat. No. 6,941,172 B2, issued to Zvi Nachum, a “Method and device for restoring kidney function using electromagnetic stimulation” is disclosed. The method of restoring kidney function includes the steps of: (a) providing a device including: a conducting coil, and a signal generator for providing a plurality of electrical impulses to the coil; (b) disposing the conducting coil proximate to a kidney of a patient, and (c) delivering the electrical impulses conducted to the conducting coil, so as to produce an electromagnetic field, the electromagnetic field acting so as to stimulate the kidney. The inventor states that this method is applicable only to the cases of acute kidney failure, mainly for traumatic ones, not to CKD: “The device and method of the present invention appear to be most effective in treating kidney failure due to trauma.
Kidney failure due to trauma is acute, and is generally reversible, at least during the initial stages. Without wishing to be limited by theory, it is believed that a static charge builds up within the tissues of the kidney, for reasons that are not yet fully understood. This static charge inhibits proper functioning of the kidney. As long as no significant irreversible damage has been caused to the kidney, the kidney can be stimulated into regaining normal performance by clearing the static charge within the tissues of the kidney by application of an electromagnetic field using the device and method of the present invention”.
It should be mentioned that it is widely accepted in the art that the biologically active component of electromagnetic stimulation is the electric field produced by the changing magnetic field. In the Nachum patent mentioned above, there is no disclosure of the magnitude of the electric field applied to the kidney. But from the time dependency of the magnetic field which is described in the patent, it can be easy estimated that the electric field was about 1 μV/cm or less. This level of electric field is, probably, enough to change the pathologic charging in the kidney caused by trauma, but it is not enough to up-regulate the A2a receptors on kidney cells. In other words, the disclosed Nachum method, developed for restoration of kidney function of a traumatized kidney, cannot be effective in preservation of kidney function deteriorating due to chronic inflammation of kidney. In particular, Nachum makes an explicit “distinction between acute and chronic renal failure is of cardinal importance” as explained in Column 1 starting at line 19 of his patent. Chronic kidney disease and acute renal failure are clearly two different diseases.
Therefore, there continues to be a need for devices and methods for treating chronic kidney diseases.